Asian Journal of Andrology (2015) 17, 298–303 © 2015 AJA, SIMM & SJTU. All rights reserved 1008-682X www.asiaandro.com; www.ajandrology.com
Open Access
Semen Analysis
ORIGINAL ARTICLE
The reference values for semen parameters of 1213 fertile men in Guangdong Province in China Yun‑Ge Tang1,2,3, Li‑Xin Tang1,2,3, Qi‑Ling Wang1,2,3, Ge Song1,4, Yan‑Jia Jiang1,2, Shun‑Mei Deng1,2, Fang Jiang1,2,3, Wei‑Bing Qin1,2,3 Semen samples were collected from 1213 fertile men whose partners had a time‑to‑pregnancy (TTP) ≤12 months in Guangdong Province in Southern China, and semen parameters including semen volume, sperm concentration, total counts, motility, and morphology were evaluated according to the World Health Organization (WHO) 2010 guideline. All semen parameters analyzed were normal in ~62.2% of the total samples, whereas ~37.8% showed at least one of the semen parameters below normal threshold values. The fifth centiles (with 95% confidence intervals) were 1.3 (1.2–1.5) ml for semen volume, 20 × 106 (18×106– 20×106) ml−1 for sperm concentration, 40 × 106 (38×106–44×106) per ejaculate for total sperm counts, 48% (47%–53%) for vitality, 39% (36%–43%) for total motility, 25% (23%–27%) for sperm progressive motility, 5.0% (4%–5%) for normal morphology. The pH values ranged from 7.2 to 8.0 with the mean ± standard deviation at 7.32 ± 0.17. No effects of age and body mass index were found on semen parameters. Occupation, smoking and alcohol abuse, varicocele appeared to decrease semen quality. Sperm concentration, but not sperm morphology, is positively correlated with TTP, whereas vitality is negatively correlated with TTP. Our study provides the latest reference values for the semen parameters of Chinese fertile men in Guangdong Province, which are close to those described in the new WHO guidelines (5th Edition). Asian Journal of Andrology (2015) 17, 298–303; doi: 10.4103/1008-682X.143251; published online: 18 November 2014 Keywords: Chinese fertile men; semen parameters; semen quality; time‑to‑pregnancy
INTRODUCTION Semen quality has been commonly regarded as a measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology, and pregnancy risk assessments.1 Since the publication of a meta‑analysis showing a major decline in sperm counts over a period of 50 years after the second world war,2 many retrospective, comparative studies on semen quality around the world have been published over the past decade. Several reports suggested a decline,3–8 whereas others showed no significant changes in semen quality in men.9–14 Similar debatable data on the semen quality in China have also been published; by reviewing 115 reports published between 1985 and 2009, Huang et al.14 analyzed the data from 23 126 healthy Chinese subjects and demonstrated a possible decline in sperm concentration. In contrast, Wen et al.15 reported no evidence of decline in sperm concentration during a period of 18 years in Guangdong Province in Southern China. Semen quality has been considered as one of the most sensitive indicators of the adverse effects of environmental pollution.16 In addition to physical environments, semen quality is also affected by other factors, such as age,17,18 occupation,19 cigarette smoking,20,21 and lifestyle.22,23 Most of the previous studies used less defined semen samples from laboratories in fertility clinics, whereas two recent studies from China investigated well‑defined groups representative of the population of healthy young men.24,25 Other deficiencies of previous
studies, especially those published in the earlier years,26–28 include the lack of comparable laboratory techniques and inappropriate statistical analyses. The present study was aimed at a comprehensive assessment of the current status of semen parameters in Chinese fertile men whose partners had time‑to‑pregnancy (TTP) ≤12 months in Guangdong area according to the strict guideline of World Health Organization (WHO) laboratory manual for the examination and processing of human semen (2010, 5th Edition). MATERIALS AND METHODS Subjects Husbands of pregnant women who had TTP ≤ 12 months from Heyuan, Jiangmen, Yangjiang, Zhanjiang, and Qingyuan areas in Guangdong province were invited to participate in our study from October 2010 to September 2012 by the local Family Planning Network. The total of 1258 fertile men were invited to participate and 1213 (96.4%) agreed. The eligibility criteria for the male participants were as follows: age 20–45 years at the time of invitation, residence of the local area near the hospital where he was recruited. In addition, the woman’s current pregnancy had to be achieved by normal sexual intercourse rather than fertility treatment. Subjects with the following conditions were excluded: epididymitis, cryptorchidism, orchitis, genital tract surgery, chemotherapy, radiotherapy or chronic illness, previous
Key Laboratory of Male Reproductive and Genetics, National Health and Family Planning Commission, Guangzhou 510600, China; 2Department of Male Reproduction, Family Planning Research Institute of Guangdong, Guangzhou 510600, China; 3Human Sperm Bank of Guangdong Province, Guanzghou 510600, China; 4Reproductive Medicine Center, Family Planning Research Institute of Guangdong, Guangzhou 510600, China. Correspondence: Dr. YG Tang ([email protected]) or Dr. WB Qin ([email protected]) Received: 03 April 2014; Revised: 09 June 2014; Accepted: 22 August 2014 1
Semen quality of fertile men in China YG Tang et al 299
treatment for infertility or reduced fertility, and unwanted pregnancy or prolonged TTP. Questionnaires Both the men and their pregnant wives were asked to complete a questionnaire. The questionnaire included detailed information on demography, education, lifestyle, occupational exposure, reproductive history, consumption of tobacco and alcohol, and previous or current diseases. The participants were informed of the purpose of the study, and possible benefits and risks of participating in the study. All participants were asked to sign an informed consent form if they agreed to take part in this study. The protocol for the use of human subjects was approved by the Human Subject Ethics Committee of the Research Institute of Family Planning of Guangdong Province. Physical examination Physical examinations of all subjects were performed by the same two experienced andrologists. The results of examinations were recorded in a standard form. Secondary sexual characteristics and the possible presence of a varicocele, a hydrocele, the location of the testis in the scrotum, and the consistency of the testis and epididymis were examined to exclude the subjects with reproductive or urological diseases. Weight (kg) and height (cm) were measured using the same calibrated instrument in each center. Semen samples All subjects were asked to abstain from ejaculation for a period of 2–7 days. The period of ejaculation abstinence was calculated as the time between the current and previous ejaculations as reported by the subjects. The exact duration (in days) of abstinence was documented for each participant. The participants collected the ejaculates by masturbation at the local Family Planning Institutions into a sterile plastic container (MaleFree™SCD‑II, BRED Life Science, Shenzhen, China) and immediately delivered the sample to a laboratory in the same building. The semen samples were marked with an anonymous serial number, and were then incubated in a water‑bath at 37°C until analysis. The semen volume was measured after samples were liquefied. All samples were analyzed within 60 min after collection. Sperm motility was analyzed using a computer‑assisted semen analysis (CASA) system (SCA2000, Microptic, Barcelona, Spain). According to the WHO reference, percent of motile sperm was scored using category A (rapid progressive motility [PR]), category B (slow PR), category C (nonprogressive motility [NP]), and category D (immotility). Two types of motility were evaluated in the present study: PR and NP. Other CASA parameters analyzed included curvilinear velocity (VCL), straight‑line velocity (VSL), average path velocity, beat cross frequency, and amplitude of lateral head displacement. The calculated parameters were linearity (LIN = VSL/VCL × 100) and straightness (STR = VSL/VCL). To avoid using CASA parameters that reflect different aspects of the same movement as described in previous studies,29,30 we selected VSL (for progression), VCL (for vigor), and LIN (for swimming pattern) for statistical analyses. For morphological evaluation, semen smears were fixed with a mixture of absolute alcohol and acetone (at a ratio of 2:1), followed by staining using the modified Shorr solution. Assessment of sperm morphology was conducted according to the criteria published by the WHO, 2010, the 5th edition. Quality control of semen analysis To minimize variations in the assessment of sperm characteristics, samples from all five centers were analyzed by the same two well‑trained
technicians using the same instruments and methods. One technician evaluated appearance, viscosity, liquefaction time, semen volume, while the other measured sperm concentration, motility, and morphology. The two technicians participated in the continuous quality control system, an external quality control system established based on the WHO guidelines, supervised by the Quality Control Center of Guangdong Province. Semen quality was evaluated based on the recommendations by the WHO, 2010. Statistical analyses Because semen parameters follow markedly nonnormal distributions, unadjusted percentiles, and medians were calculated for the semen parameters. Percentages coincident with the criteria of WHO (2010) were also calculated. The data were also summarized using median, 5th percentiles, and were stratified by age, body mass index (BMI), occupation and lifestyle. Kruskal–Wallis analysis of variance, a nonparametric test, was used to compare medians between groups. A step‑wise multiple regression analysis was used to determine which variables are the strongest independent correlates of TTP. A generalized linear model was used to examine the independent effect of risk factors on semen parameters. A full model that included all risk factors examined in the final regression was used. Independent variables entered into the regression model as dummy variables were as follows: (i) age: ≥35, 30–35, 25–30, and 25.0,
Open Access
Semen Analysis
ORIGINAL ARTICLE
The reference values for semen parameters of 1213 fertile men in Guangdong Province in China Yun‑Ge Tang1,2,3, Li‑Xin Tang1,2,3, Qi‑Ling Wang1,2,3, Ge Song1,4, Yan‑Jia Jiang1,2, Shun‑Mei Deng1,2, Fang Jiang1,2,3, Wei‑Bing Qin1,2,3 Semen samples were collected from 1213 fertile men whose partners had a time‑to‑pregnancy (TTP) ≤12 months in Guangdong Province in Southern China, and semen parameters including semen volume, sperm concentration, total counts, motility, and morphology were evaluated according to the World Health Organization (WHO) 2010 guideline. All semen parameters analyzed were normal in ~62.2% of the total samples, whereas ~37.8% showed at least one of the semen parameters below normal threshold values. The fifth centiles (with 95% confidence intervals) were 1.3 (1.2–1.5) ml for semen volume, 20 × 106 (18×106– 20×106) ml−1 for sperm concentration, 40 × 106 (38×106–44×106) per ejaculate for total sperm counts, 48% (47%–53%) for vitality, 39% (36%–43%) for total motility, 25% (23%–27%) for sperm progressive motility, 5.0% (4%–5%) for normal morphology. The pH values ranged from 7.2 to 8.0 with the mean ± standard deviation at 7.32 ± 0.17. No effects of age and body mass index were found on semen parameters. Occupation, smoking and alcohol abuse, varicocele appeared to decrease semen quality. Sperm concentration, but not sperm morphology, is positively correlated with TTP, whereas vitality is negatively correlated with TTP. Our study provides the latest reference values for the semen parameters of Chinese fertile men in Guangdong Province, which are close to those described in the new WHO guidelines (5th Edition). Asian Journal of Andrology (2015) 17, 298–303; doi: 10.4103/1008-682X.143251; published online: 18 November 2014 Keywords: Chinese fertile men; semen parameters; semen quality; time‑to‑pregnancy
INTRODUCTION Semen quality has been commonly regarded as a measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology, and pregnancy risk assessments.1 Since the publication of a meta‑analysis showing a major decline in sperm counts over a period of 50 years after the second world war,2 many retrospective, comparative studies on semen quality around the world have been published over the past decade. Several reports suggested a decline,3–8 whereas others showed no significant changes in semen quality in men.9–14 Similar debatable data on the semen quality in China have also been published; by reviewing 115 reports published between 1985 and 2009, Huang et al.14 analyzed the data from 23 126 healthy Chinese subjects and demonstrated a possible decline in sperm concentration. In contrast, Wen et al.15 reported no evidence of decline in sperm concentration during a period of 18 years in Guangdong Province in Southern China. Semen quality has been considered as one of the most sensitive indicators of the adverse effects of environmental pollution.16 In addition to physical environments, semen quality is also affected by other factors, such as age,17,18 occupation,19 cigarette smoking,20,21 and lifestyle.22,23 Most of the previous studies used less defined semen samples from laboratories in fertility clinics, whereas two recent studies from China investigated well‑defined groups representative of the population of healthy young men.24,25 Other deficiencies of previous
studies, especially those published in the earlier years,26–28 include the lack of comparable laboratory techniques and inappropriate statistical analyses. The present study was aimed at a comprehensive assessment of the current status of semen parameters in Chinese fertile men whose partners had time‑to‑pregnancy (TTP) ≤12 months in Guangdong area according to the strict guideline of World Health Organization (WHO) laboratory manual for the examination and processing of human semen (2010, 5th Edition). MATERIALS AND METHODS Subjects Husbands of pregnant women who had TTP ≤ 12 months from Heyuan, Jiangmen, Yangjiang, Zhanjiang, and Qingyuan areas in Guangdong province were invited to participate in our study from October 2010 to September 2012 by the local Family Planning Network. The total of 1258 fertile men were invited to participate and 1213 (96.4%) agreed. The eligibility criteria for the male participants were as follows: age 20–45 years at the time of invitation, residence of the local area near the hospital where he was recruited. In addition, the woman’s current pregnancy had to be achieved by normal sexual intercourse rather than fertility treatment. Subjects with the following conditions were excluded: epididymitis, cryptorchidism, orchitis, genital tract surgery, chemotherapy, radiotherapy or chronic illness, previous
Key Laboratory of Male Reproductive and Genetics, National Health and Family Planning Commission, Guangzhou 510600, China; 2Department of Male Reproduction, Family Planning Research Institute of Guangdong, Guangzhou 510600, China; 3Human Sperm Bank of Guangdong Province, Guanzghou 510600, China; 4Reproductive Medicine Center, Family Planning Research Institute of Guangdong, Guangzhou 510600, China. Correspondence: Dr. YG Tang ([email protected]) or Dr. WB Qin ([email protected]) Received: 03 April 2014; Revised: 09 June 2014; Accepted: 22 August 2014 1
Semen quality of fertile men in China YG Tang et al 299
treatment for infertility or reduced fertility, and unwanted pregnancy or prolonged TTP. Questionnaires Both the men and their pregnant wives were asked to complete a questionnaire. The questionnaire included detailed information on demography, education, lifestyle, occupational exposure, reproductive history, consumption of tobacco and alcohol, and previous or current diseases. The participants were informed of the purpose of the study, and possible benefits and risks of participating in the study. All participants were asked to sign an informed consent form if they agreed to take part in this study. The protocol for the use of human subjects was approved by the Human Subject Ethics Committee of the Research Institute of Family Planning of Guangdong Province. Physical examination Physical examinations of all subjects were performed by the same two experienced andrologists. The results of examinations were recorded in a standard form. Secondary sexual characteristics and the possible presence of a varicocele, a hydrocele, the location of the testis in the scrotum, and the consistency of the testis and epididymis were examined to exclude the subjects with reproductive or urological diseases. Weight (kg) and height (cm) were measured using the same calibrated instrument in each center. Semen samples All subjects were asked to abstain from ejaculation for a period of 2–7 days. The period of ejaculation abstinence was calculated as the time between the current and previous ejaculations as reported by the subjects. The exact duration (in days) of abstinence was documented for each participant. The participants collected the ejaculates by masturbation at the local Family Planning Institutions into a sterile plastic container (MaleFree™SCD‑II, BRED Life Science, Shenzhen, China) and immediately delivered the sample to a laboratory in the same building. The semen samples were marked with an anonymous serial number, and were then incubated in a water‑bath at 37°C until analysis. The semen volume was measured after samples were liquefied. All samples were analyzed within 60 min after collection. Sperm motility was analyzed using a computer‑assisted semen analysis (CASA) system (SCA2000, Microptic, Barcelona, Spain). According to the WHO reference, percent of motile sperm was scored using category A (rapid progressive motility [PR]), category B (slow PR), category C (nonprogressive motility [NP]), and category D (immotility). Two types of motility were evaluated in the present study: PR and NP. Other CASA parameters analyzed included curvilinear velocity (VCL), straight‑line velocity (VSL), average path velocity, beat cross frequency, and amplitude of lateral head displacement. The calculated parameters were linearity (LIN = VSL/VCL × 100) and straightness (STR = VSL/VCL). To avoid using CASA parameters that reflect different aspects of the same movement as described in previous studies,29,30 we selected VSL (for progression), VCL (for vigor), and LIN (for swimming pattern) for statistical analyses. For morphological evaluation, semen smears were fixed with a mixture of absolute alcohol and acetone (at a ratio of 2:1), followed by staining using the modified Shorr solution. Assessment of sperm morphology was conducted according to the criteria published by the WHO, 2010, the 5th edition. Quality control of semen analysis To minimize variations in the assessment of sperm characteristics, samples from all five centers were analyzed by the same two well‑trained
technicians using the same instruments and methods. One technician evaluated appearance, viscosity, liquefaction time, semen volume, while the other measured sperm concentration, motility, and morphology. The two technicians participated in the continuous quality control system, an external quality control system established based on the WHO guidelines, supervised by the Quality Control Center of Guangdong Province. Semen quality was evaluated based on the recommendations by the WHO, 2010. Statistical analyses Because semen parameters follow markedly nonnormal distributions, unadjusted percentiles, and medians were calculated for the semen parameters. Percentages coincident with the criteria of WHO (2010) were also calculated. The data were also summarized using median, 5th percentiles, and were stratified by age, body mass index (BMI), occupation and lifestyle. Kruskal–Wallis analysis of variance, a nonparametric test, was used to compare medians between groups. A step‑wise multiple regression analysis was used to determine which variables are the strongest independent correlates of TTP. A generalized linear model was used to examine the independent effect of risk factors on semen parameters. A full model that included all risk factors examined in the final regression was used. Independent variables entered into the regression model as dummy variables were as follows: (i) age: ≥35, 30–35, 25–30, and 25.0,
